The expression of functional antibodies and antibody fragments in E. coli is known in the prior art, but these methods require the use of signal sequences which direct polypeptide transport into the periplasm. When expression takes place in the E. coli periplasm, the expression yields are in the range of a few μg per liter of culture medium (Ayala et al., Bio Techniques 13, pp. 790-799, 1992). In addition, refolding experiments are often required in order to obtain functionally active antibody fragments (such as Fab) or antigen-binding regions (such as a single chain Fv(sFv)). There is a need therefore, to develop improved methods for expressing functionally active antibodies and antibody fragments. The prior art does not teach recombinant production of antibodies or antibody fragments which can be isolated from the cytoplasm in functional form. Such molecules would be useful in the production of therapeutic agents.
Bagshawe describes a method for generating cytotoxic agents that are directed towards cancer sites, termed Antibody Directed Enzyme Prodrug Therapy (ADEPT). Bagshawe, Br. J. Cancer, vol. 60, pp. 275-281, 1989. Using ADEPT, an antibody or antibody fragment that specifically binds to a cancer cell is fused to an enzyme that is capable of converting a non-toxic drug into a toxic drug. Only those cells to which the fusion protein is bound will be killed upon administration of the precursor of the toxic drug.
The β-glucuronidase of Escherichia coli has been well characterized biochemically and genetically. The gene (uid A) has been cloned by Jefferson et al. (PNAS vol. 83, pp. 8447-8451, 1986) and employed as a reporter gene for heterologous control regions.
β-Glucuronidase (β-D-glucuronoside glucuronosohydrolase, E.C. 3.2.1.31) is an acid hydrolase which catalyzes the cleavage of β-glucuronides. As a result of the mammalian glucuronidases having been intensively investigated, a variety of substances are available for histological, spectrophotometric and fluorometric analyses. This enzyme has gained new, additional importance in its use for fusion proteins for targeted tumor therapy. In this connection, human glucuronidase is used in the form of a fusion protein which contains antibodies/antibody fragments or antigen-binding regions (Bosslet et al., Br. J, Cancer, 65, 234-238, 1992). As an alternative to the human enzyme, it is also possible to use the homologous E. coli β-glucuronidase. One of the advantages of the E. coli β-glucuronidase is that its catalytic activity at physiological pH is significantly higher than that of the human β-glucuronidase.
In the past, it has only been possible to express antibody fragment-enzyme fusion molecules periplasmically in E. coli. The enzyme moiety which is used in this context is therefore always composed of periplasmic E. coli enzymes such as β-lactamase (Goshorn et al., Canc. Res. 53, 2123-2117, 1993).
An E. coli strain which is deficient in thioredoxin reductase (TRR), for example the strain AD 494, is capable of forming disulfide bridges in the cytoplasm and thus enzymes which are naturally secretory, for example alkaline phosphatase, can be expressed intracellularly See Derman et al., Science, 262:1744-1747, 1993. Derman describes the selection and isolation of TRR-deficient E. coli mutants.
The prior art does not teach expression of an antibody fragment-enzyme fusion molecule using a cytoplasmic E. coli enzyme, such as β-glucuronidase., which is functionally active—i.e., which retains both enzymatic activity and antigen-binding ability of the antibody moiety. As a rule, functionally active expression of most antibodies or antibody fragment molecules requires defined signal sequences for exporting the expressed molecules via the endoplasmic reticulum into the culture medium (animal cells and yeast) or into the periplasm (E. coli). It is only in the endoplasmic reticulum or in the periplasm that the necessary oxidative conditions pertain for forming the disulfide bridges which are important for functional activity. In addition, the secretory synthesis route is often crucial for the correct three-dimensional folding of the expressed protein.